Rituximab in the treatment of autoimmune haematological disorders.

Current treatment regimens for haematological autoimmune diseases are relatively non-selective and are often associated with considerable toxicity. Recently, it has become clear that B cells play a key role in both the development and perpetuation of autoimmunity, suggesting that B-cell depletion could be a valuable treatment approach for patients with autoimmune diseases. This article reviews data supporting the use of rituximab--an anti-CD20 monoclonal antibody that specifically depletes B cells--in four key autoimmune haematological disorders: idiopathic thrombocytopenic purpura (ITP); autoimmune haemolytic anaemia (AIHA); acquired haemophilia; and thrombotic thrombocytopenic purpura (TTP). Although treatment of ITP, AIHA, acquired haemophilia and TTP with rituximab is still relatively uncommon, results from case series and small phase II trials indicate that patients of all ages can respond to rituximab, irrespective of the number or type of prior treatments that they have received. Moreover, patients with these diseases receiving rituximab experienced predominantly mild adverse events, with only a few serious adverse events reported. These data suggest that rituximab provides an effective and well-tolerated alternative treatment option for patients with ITP, AIHA, acquired haemophilia and TTP, many of whom have limited treatment choices.

Plasmapheresis and steroid treatment of levamisole-induced vasculopathy and associated skin necrosis in crack/cocaine users.

BACKGROUND: Levamisole was removed from the market due to complications of agranulocytosis and skin necrosis. Levamisole has been reported in a high proportion of seized cocaine in North America and has been associated with multiple cases of skin necrosis. OBJECTIVE: We report three cases of levamisole/cocaine-induced skin necrosis who responded to treatment with plasmapheresis and immunosuppression. RESULTS: Three patients presented with painful necrotic skin lesions on the ears, cheeks, breasts, and buttocks. The extremities were involved in two patients and the upper respiratory tract mucosa in one patient. All had markers of immune activation, with elevated C-reactive protein, antinuclear antibody, and perinuclear antineutrophil cytoplasmic antibody. Skin biopsy in all cases revealed a mixed pattern of thrombosis and vasculitis within dermal vessels, with overlying ischemic ulceration of skin and soft tissues. One patient required extensive débridement of the skin and soft tissue of the calves and also had respiratory involvement. All patients were treated with plasmapheresis and immunosuppression with rapid stabilization and/or improvement of the lesions. CONCLUSION: Levamisole is frequently added to crack/cocaine; we report three patients who developed vascular lesions and skin necrosis after using cocaine/levamisole. These improved with plasmapheresis and immunosuppression as well as abstention from the drugs; one patient with severe disease required débridement and skin grafting.

Sepsis- and endotoxemia-generated cytokines do not trigger activation of human platelets.

OBJECTIVE: To analyze the effect of cytokines generated in sepsis and endotoxemia (tumor necrosis factor [TNF]-alpha and interleukins [IL]-1beta, -6, and -8) on activation of human platelets and to study the effect of cytokines on platelet activation in the presence of alpha-thrombin, a potent inducer of coagulation and platelet activation generated in sepsis and endotoxemia. DESIGN: flow cytometric study of platelet activation induced by cytokines and/or thrombin in the whole blood and platelet-rich plasma (PRP) of healthy volunteers. SETTING: Research laboratory in a Canadian hospital. SUBJECTS: Nine healthy volunteers recruited from laboratory staff. MEASUREMENTS AND MAIN RESULTS: Venous blood samples were obtained into acid-citrate-dextrose anticoagulant. Whole blood and PRP were diluted with appropriate buffer optimized for analyzing platelet activation by flow cytometry. TNF-alpha, IL-1beta, IL-6, and IL-8 were added to blood or PRP in concentrations ranging from 1 to 100 ng/mL and incubated for 15 mins at 37 degrees C in the presence or absence of a submaximal concentration of human alpha-thrombin (0.025 units/mL). Samples were stained with fluorescent antibodies against markers of platelet activation (P-selectin [CD62], lysosomal protein [CD63], and fibrinogen and von Willebrand factor receptors [CD41 and CD42b, respectively]) and analyzed by flow cytometry. The data obtained show that none of these cytokines trigger activation of resting platelets in whole blood or PRP and do not modulate the effect of thrombin on platelet activation as measured by quantitation of CD62, CD63, and CD42b markers on the platelet surface. CONCLUSIONS: Cytokines TNF-alpha, IL-1beta, IL-6, and IL-8, which are extensively produced in sepsis and endotoxemia, do not trigger activation of resting human platelets directly or indirectly by mediating processes in white or red blood cells. The cytokines did not affect thrombin-induced platelet activation.

Hemolink, an o-raffinose cross-linked haemoglobin-based oxygen carrier, does not affect activation and function of human platelets in whole blood in vitro.

Haemoglobin-based oxygen carriers (HBOCs) are anticipated to be safe and efficient alternatives to RBC transfusions. Haemoglobin (Hb) raffimer (Hemolink; Hemosol, Toronto, ON, Canada) is polymerized human Hb, cross-linked with o-raffinose. As administration of cell-free Hb may affect blood cells and tissues, this study was focused on evaluating effects of Hb raffimer on human platelets in whole blood in vitro. Citrated blood from healthy donors was incubated with Hb raffimer to achieve raffimer concentrations of 2-50 vol percentage (2-50 g/l). Platelet activation, phosphatidylserine exposure and microparticle generation were measured by flow cytometry. Aperture closure time on collagen/ADP- and collagen/epinephrine-coated membranes was determined by a platelet function analyser (PFA-100). We found that addition of Hb raffimer to blood samples up to 50 vol % did not affect human platelets as measured by various markers of platelet activation (CD42b, CD41, PAC-1, CD62, CD63), procoagulant activity (annexin V) and microparticle formation; differences between Hb raffimer- and lactated Ringer's-diluted blood were not significant. Similarly, no adverse effect of Hb raffimer on closure time was observed at concentrations up to 50 vol %, in comparison with Ringer's solution. These data indicate that exposure of human blood to high concentrations of Hb raffimer in vitro did not cause platelet activation nor affect platelet function.

Role of platelet surface glycoprotein Ibalpha and P-selectin in the clearance of transfused platelet concentrates.

BACKGROUND: Role of P-selectin (CD62) and glycoprotein (GP) Ibalpha in posttransfusion clearance of platelet concentrates (PCs) is unclear. STUDY DESIGN AND METHODS: Platelet (PLT) activation in vitro was determined by flow cytometry using anti-CD62 and anti-GPIbalpha. PC clearance in vivo was evaluated in an animal model using rabbits with an inhibited reticuloendothelial system, as measured by 0.5-hour (R(0.5)), 24-hour (R(24)), and total (R( summation operator )) PLT recoveries, and survival time (ST). Correlations were analyzed between in vitro assays of PLT activation and in vivo clearance of conventional (Days 2-5), outdated (Days 7-8), and refrigerated PCs. RESULTS: Binding of anti-CD62 to the PLT surface was significantly increased and of anti-GPIbalpha decreased in outdated and refrigerated PCs compared to conventional PCs. Negative correlation was observed between in vitro anti-CD62 binding and the fast (R(0.5)) PLT clearance, but not with delayed (R(24) and ST) clearance. In contrast, anti-GPIbalpha binding showed positive correlations with delayed but not with fast PLT clearance. Overall (R( summation operator )) clearance correlated better with anti-GPIbalpha than with anti-CD62 binding. CD62 density on the PLT surface was decreased after PC transfusion, whereas GPIbalpha density remained unchanged. CONCLUSION: These data suggest that CD62 exposure on the PLT surface during PC storage triggers fast CD62-mediated PC clearance, whereas in vitro GPIbalpha changes are involved in delayed GPIbalpha-mediated PC clearance.

A rabbit model for monitoring in vivo viability of human platelet concentrates using flow cytometry.

BACKGROUND: Viability in vivo of novel platelet components cannot be readily determined in human transfusions. Elaboration of valid animal models may be useful for this purpose. STUDY DESIGN AND METHODS: Viability of platelet concentrates (PCs) WBC reduced before storage was determined by flow cytometry in rabbits whose reticuloendothelial system was inhibited by ethyl palmitate administration. PCs stored at 22 degrees C for 2 and 5 days (D2- and D5-PCs) or refrigerated PCs (3-6 days at 22 degrees C plus 1-4 days at 4 degrees C, RF-PCs) were transfused into rabbits. Five parameters of PC viability in vivo were calculated from human platelet survival curves: survival time, recovery 0.5 and 24 hours after transfusion (R0.5, R24), maximal recovery (Rmax), and total recovery for 0 to 24 hours (RSigma). RESULTS: No differences in viability of D2- and D5-PCs were found. In contrast, viability of RF-PCs was significantly lower than that of D2-PCs, as was revealed with diverse sensitivity by four parameters: RSigma > R24 > R0.5=survival time (p < 0.001, p < 0.01, and p < 0.05, respectively). CONCLUSION: The rabbit model elaborated is sufficiently sensitive to reveal differences in human platelet viability in vivo between conventional and cold-damaged PCs. It may be useful for comparing viability of different platelet components that cannot be readily tested in human transfusions.

Pathologic high shear stress induces apoptosis events in human platelets.

Recently, it has been discovered that apoptosis of anucleate platelets can be induced by chemical agonists. Other studies demonstrated that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analyzed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer, we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arteriole levels (10-44 dyn/cm2) to pathologic high levels (117-388 dyn/cm2) occurring in stenotic vessels. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIbalpha downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation, whereas physiological shear stresses are not effective. This novel finding suggests that shear-induced platelet apoptosis can be mediated by mechanoreceptors, does not require nuclear participation, and may affect platelet clearance.